The recent discovery that de novo zygotic mutations in the form of CNVs and point mutations make major contributions to neuropsychiatric diseases such as schizophrenia and autism begs the question as to the degree to which de novo post-zygotic mosaic mutations also contribute to disease. In this model, a mutation that occurs post-zygotically can seed some percentage of cells in the brain, and is sufficient to lead to neuronal dysfunction and disease. The approach of sequencing only non-neural tissues such as blood may underpower the detection of mosaicism, because mutations may be restricted to neural tissue. This proposal brings together a highly productive and collaborative team, in which each member contributes a special resource to make this effort truly unique. Gleeson and Mathern recently identified among the first de novo somatic mutations in the developing brain in the condition known as `hemimegalencephaly' (HME), a catastrophic neuropsychiatric condition associated with focal cortical disorganization (FCD). By comparing DNA from diseased brain vs. blood, we identified de novo somatic mutations in PIK3CA, AKT3 and MTOR, part of the mTOR pathway, in as few as 8% of brain cells, resulting in perturbations in an entire cerebral hemisphere. Courchesne and Roy recently identified focal patches of abnormal laminar cytoarchitecture in frontal and temporal cortex in the majority of available brain samples of children studied with autism (ASD), and we suggest focal patches akin to FCD may have similar mutations and contribute to disease. The goal of this application is to extend the discovery of mosaicism in patients with epilepsy and autism in which neurohistopathological evidence points to FCD, by sequencing dysplasias compared with adjacent normal tissue and/or blood at the DNA and RNA level at the single-cell level. We aim to uncover key sets of genes, in which specific de novo mutations, in specific locations, at specific mosaicism levels, is sufficient to produce clinically defined disease. We will combine next-generation sequencing of FCD brain from patients with neuropsychiatric disease with advanced bioinformatics, single-cell sequencing, complete clinical correlated neuroanatomy and mouse modeling. We will: 1] Test for de novo somatic mutations in a retrospective and prospective cohort of FCD presenting with autism or epilepsy. 2] Correlate genetic disease burden with clinical, imaging, histopathological and single-cell sequencing findings. 3] Test mechanisms by which uncovered de novo mutations alter progenitor cell functions in mammalian cerebral cortex.